Cell locomotion is critical to development, wound healing and tissue remodeling, and a central problem during metastasis in cancer. Thus, it is important to gain an understanding of cell motility at the basic level. The cytoskeletal polymers, microtubules and filamentous actin (f-actin) are required for cell motility. Polarized f-actin assembly and movement is thought to drive cell motility, while microtubules are believed to spatially direct sites of f-actin assembly. There is evidence that microtubules may achieve this by activation of cellular signaling via small GTPases of the Rho family. However, the organization and dynamics of microtubules and how they are affected by actin is not thoroughly characterized, and the molecules involved in mediating their interactions during cell motility are unknown. I approach these problems at 3 levels: First, by examining microtubules and f-actin dynamics in living cells using multimode microscopy, by exploring with both microscopy and biochemistry microtubule/f-actin interactions in a cellular extract model systems, and finally by examining the biochemical interactions of purified components of the motile machinery in vitro. The combination of unique microscopy methods and molecular biochemistry is a novel means of carefully characterizing cellular phenomenon and then going directly to the test tube test hypotheses about the molecular mechanisms underlying the phenomena. In this proposal, I describe three specific aims to shed light on our understanding of microtubule/f- actin interactions in cell motility. 1. To develop new microscopy methods for the simultaneous visualization and analysis of f-actin and microtubule dynamics and to carefully analyze microtubule/actin interactions in living migrating cells. 2. To biochemically and microscopically probe the molecular mechanism dynamic microtubule/actin interactions in Xenopus egg extracts. 3. To biochemically analyze microtubule mediation of Rho family signaling and study the mechanism by which growing microtubules activate Rac1 during cell motility. The results of these studies will greatly enhance our understanding of the mechanisms and mechanics of cell locomotion and will provide new methods for analyzing protein dynamics in living cells.